A major determinant of CD4+ T cell activation and response is the strength of signal mediated by the trimolecular MHC-peptide-TCR interaction. This strength of signal is dependent on the density and duration of trimolecular interactions, and can be measured in terms of "functional avidity" for individual T cells. New technologies using MHC tetramers are now available to quantify functional avidity in a polyclonal T cell population, and we propose to use these techniques to evaluate the antigen-specific CD4+ T cell response in autoimmune diabetes. We hypothesize that high avidity recognition of multiple islet autoantigens correlates with disease progression or earlier disease onset. We predict that epitope spreading and high avidity responses fail to occur in subjects with non-progressive forms of islet autoimmunity, such as subjects with only a single islet autoantibody. At-risk and LADA cohorts in this program project will be compared to T1D subjects, and integrated with the autoantibody progression studies in Project 2. we will also test the hypothesis that autoreactive T cells may persist by becoming refractory to down-regulation, through the analysis of tetramer-sorted cells challenged with a variety of costimulatory or regulatory signals.